Interpretive Summary: Water-deficit stresses preferentially reduce shoot growth, thereby disrupting the flow of carbohydrates from source leaves to the developing sinks. Here, we use a novel stress bioassay to dissect responses of field and greenhouse-grown cotton (Gossypium hirsutum L.) source leaves to water-deficit stresses. The research described in this study evaluated the ability of source leaf tissues harvested at sunrise from well watered and water-deficit stressed cotton to withstand prolonged respiratory demands before experiencing complete tissue death. The working hypothesis was that overnight mobilization of stored photosynthate from source leaf tissues of rapidly growing non-stressed cotton would result in fewer reserves at sunrise then leaves from water-deficit stressed cotton experiencing restricted shoot growth. This differential in stored photosynthate at dawn would predisposed the non-stressed leaf tissue to succumb more rapidly then the water-deficit stressed source tissues that contained more stored photosynthate. Specifically, we have developed a bioassay that identifies source leaf responses to stress-induced cessation of shoot growth, and allows scientists to evaluate 200-300 samples at a time.

Technical Abstract:
Water-deficit stresses preferentially reduce shoot growth, thereby disrupting the flow of carbohydrates from source leaves to the developing sinks. Here, we use a novel stress bioassay to dissect responses of field and greenhouse-grown cotton (Gossypium hirsutum L.) source leaves to water-deficit stresses. Fifth main stem leaf samples were harvested at sunrise and subjected to a prolonged elevated respiratory demand in the dark. In non-stressed plants only a small amount of starch is left at the end of the night, thereby predisposing the tissue to succumb more rapidly then the water-deficit stressed tissues that contained more stored photosynthate. Tissue death was determined initially using the cell viability stain 2,3,5-triphenyltetrazolium chloride, but was determined in subsequent experiments by monitoring the decline in chlorophyll fluorescence yield. Fluorescence yield measurements were obtained within minutes of harvesting and individual samples were monitored over the time course of the treatment. Analyses of the time course and magnitude of chlorophyll fluorescence yield decline in samples from irrigated and dryland plots permitted the detection of stress responses within 24 h of the cessation of irrigation. The rate of fluorescence yield decline during the elevated respiratory demand treatment slowed as the water-deficit stress increased. Upon irrigation, the source leaves of the water stressed plants recovered to pre-stress values within 4 days. Well-watered cotton over-expressing hsp101 had identical rates of fluorescence yield decline as non-transgenic cotton. These results suggest that the delayed decline in fluorescence yield of water stressed tissue is primarily attributed to metabolites accumulating in response to stress.